: The VP16 protein of herpes simplex virus type 1 (HSV-1) is an important model system for studying the molecular mechanisms of transcriptional activation. This virion protein potently activates transcription of the viral immediate early (IE) genes and thus triggers the cascade of viral gene expression that underlies the lytic infection cycle. VP16 represents a key regulatory step for herpesvirus infections and may therefore be an attractive target for design of novel antiviral therapeutic strategies. The long-term goal for this project is to understand the mechanism of transcriptional activation by VP16. Whereas previous project periods have (profitably) explored VP16 as a prototypical transcriptional activator through mutational, biophysical and protein interaction studies, the present proposal seeks to understand the role of VP16 in its proper biological context as an activator of IE genes during HSV infection. In the next project period, five aspects of VP16 biology will be explored. First, in yeast and in mammalian cell extracts, VP16 can interact with coactivator proteins or complexes with histone acetyltransferase activity. The lack of nucleosomes on viral DNA makes such an association rather curious. Therefore, the role of coactivator proteins in VP16 stimulation of IE gene expression will be explored. Secondly, the cellular protein GABP binds to one class of cis elements in IE gene promoters through which VP16 activates transcription. The role of GABP in VP16 activation will be defined using in vivo analysis of the protein: DNA interactions and in vitro protein:protein interactions. Third, the regulation and biological significance of VP16 phosphorylation throughout infection will be assessed using engineered mutant virus strains. Fourth, during different stages of infection, VP16 must localize to the nucleus (to activate IE expression) or elsewhere (for forming the tegument of progeny viruses). Engineered viruses bearing a VP16-GFP fusion protein will be employed to dissect the mechanisms of VP16 localization. Finally, given that the viral DNA is packaged in capsids with spermine, the potential role of spermine catabolism in expression of IE genes will be tested using in vivo radiolabeling of spermine that locates in the virion as well as using inhibitors of polyamine metabolism. These experiments will bring a greater understanding not only of VP16 as a model transcriptional activator, but also of VP16 in its biological role as a key regulator of gene expression of an important human pathogen.